Isolation and structural characterization of a polysaccharide FCAP1 from the fruit of Cornus officinalis

Article abstract of DOI:10.1016/j.carres.2010.06.009

A water-soluble polysaccharide, FCAP1, was isolated from an alkaline extract from the fruits of Cornus officinalis. Its molecular weight was 34.5 kDa. Monosaccharide composition analysis revealed that it was composed of fucose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of 0.29:0.19:1.74:1:3.30:1.10. On the basis of partial acid hydrolysis and methylation analysis, FCAP1 was shown to be a highly branched polysaccharide with a backbone of β-(1→4)-linked-glucose partially substituted at the O-6 position with xylopyranose residues. The branches were composed of (1→3)-linked-Ara, (1→4)-linked-Man, (1→4,6)-linked-Man, (1→4)-linked-Glc, and (1→2)-linked-Gal. Arabinose, fucose, and galactose were located at the terminal of the branches. The structure was further elucidated by a specific enzymatic degradation with an endo-β-(1→4)-glucanase and MALDI-TOF-MS analysis. Oligosaccharides generated from FCAP1 indicated that FCAP1 contained XXXG-type and XXG-type xyloglucan fragments.

Full text of DOI:10.1016/j.carres.2010.06.009

Carbohydrate Research 345 (2010) 1909–1913
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Isolation and structural characterization of a polysaccharide FCAP1
from the fruit of Cornus officinalis
*
Liyan Yang, Zhongfu Wang, Linjuan Huang
Life Science College, Provincial Key Laboratory of Biotechnology, Key Laboratory of Ministry of Education for Western China Resource Biology and Biotechnology,
Northwest University, Xi’an 710069, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 April 2010
Received in revised form 13 June 2010
Accepted 16 June 2010
Available online 23 June 2010
A water-soluble polysaccharide, FCAP1, was isolated from an alkaline extract from the fruits of Cornus
officinalis. Its molecular weight was 34.5 kDa. Monosaccharide composition analysis revealed that it
was composed of fucose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of
0.29:0.19:1.74:1:3.30:1.10. On the basis of partial acid hydrolysis and methylation analysis, FCAP1 was
shown to be a highly branched polysaccharide with a backbone of b-(1?4)-linked-glucose partially
substituted at the O-6 position with xylopyranose residues. The branches were composed of (1?3)-
linked-Ara, (1?4)-linked-Man, (1?4,6)-linked-Man, (1?4)-linked-Glc, and (1?2)-linked-Gal. Arabi-
nose, fucose, and galactose were located at the terminal of the branches. The structure was further
elucidated by a specific enzymatic degradation with an endo-b-(1?4)-glucanase and MALDI-TOF-MS
analysis. Oligosaccharides generated from FCAP1 indicated that FCAP1 contained XXXG-type and XXG-
type xyloglucan fragments.
Keywords:
Cornus officinalis
Polysaccharide
Structure
Xyloglucan
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
?4)-GalA-(1?2)-Rha-(1? and side chains attached to O-4 of some
rhamnose residues. The side chains contained highly branched
Cornus officinalis Sieb. et Zucc. belongs to the family Cornaceae.
The fruit has been used as a traditional herbal medicine for more
than 2000 years in China for its tonic, analgesic, and diuretic ef-
fects.1 It has also been reported to have anti-microbial, anti-inflam-
matory, anti-shock, cardiotonic, anti-oxidant, anti-aging, and
immunoregulatory activities.2
arabinan and short linear (1?3)-linked galactans. The polysaccha-
ride can be categorized to rhamnogalacturonan I (RG-I) of pectin.
In this study, we obtained a water-soluble hemicellulosic poly-
saccharide with XXXG-type and XXG-type xyloglucans by treating
the cellular debris with sodium hydroxide (5% and 10%) after water
extraction of C. officinalis. The glucan backbone is substituted at C-6
Likewise, polysaccharides isolated fromC. officinalis were shown
to have immunostimulatory,3 anti-oxidant, and anti-aging effects.4
Significant differences were reported for the monosaccharide com-
position and the chemical structure of polysaccharides extracted
from C. officinalis with hot water. For example, cornus polysaccha-
ride SZYP-2 was reported to be composed of rhamnose, arabinose,
and glucose.5 Cornus polysaccharide Co-4 was reported to be com-
posed of glucose, xylose, galactose, and fucose. Linkage analysis
of Glc (G) residues by terminal xylopyranose (X) residues, or b-
Galp-(1?2)- -Xylp (L), or -Fucp-(1?2)-b- -Galp-(1?2)-
Xylp (F) side chains leading to XXLG/XLXG, XLLG, XLFG, LGG,
XLG, XFG, and LFG xylogulcan fragments. Its isolation, homogene-
ity, molecular weight, monosaccharide composition, and structural
characterization were investigated.
D
-
-
a-D
a
-L
D
a-
D
2. Experimental
2.1. Materials
showed that Co-4 had a backbone of (1?4)-linked
dues with some branching at O-6. The branches were mainly com-
posed of (1?2)-linked -xylose, terminal xylose, terminal fucose,
and (1?2)-linked
-galactose.6
D-glucose resi-
D
D
In contrast, in our previous work,7 we characterized a polysac-
charide FCP5-A extracted with water (30 °C twice and then 80 °C
once) from the fruit ofC. officinalis. FCP5-A was a branched polysac-
charide rich in arabinose with a backbone composed of repeating
The dried pitted mature fruit of C. officinalis was purchased from
Xi’an Medicinal Materials Cooperation, PR China. It was dried at
50 °C and crushed into a powder.
2.2. Analytical methods
The carbohydrate content of the powder was determined by the
H2SO4–phenol method using glucose as a standard. Protein content
* Corresponding author. Tel.: +86 29 88303484; fax: +86 29 88373079.
E-mail address: huanglj@nwu.edu.cn (L. Huang).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.06.009

1910
L. Yang et al. / Carbohydrate Research 345 (2010) 1909–1913
was estimated by the method of Bradford using chicken egg white
albumin as standard.8 A Lambda 25 UV/VIS spectrophotometer
(Perkin Elmer instrument) was used to record the absorbance. A
UV scan in the region of 200–400 nm was performed on the spec-
trophotometer. The IR spectrum was determined using an EQUI-
NOX 55 Fourier transform infrared spectrophotometer (Bruker,
Germany). Matrix-assisted laser desorption ionization-time of
flight mass spectrum (MALDI-TOF-MS) was performed on a Shima-
dzu/Kratos (Columbia, MD) AXIMA CFR mass spectrometer
equipped with a nitrogen laser operating at 337 nm in a reflection
mode. Profiling of the molecular ions of the sample was achieved in
the positive mode using 2,5-dihydroxybenzoic acid (10 mg/mL) as
the matrix solution.
coupled with Waters Alliance 2414 refractive index detector was
used. The temperature of the column and the detector was kept
at 30 °C. The sample concentration was 0.2% (w/v), and its injection
volume was 20
phate buffer (pH 6.0) and 0.1 mol/L Na2SO4, passed through Milli-
pore filters (0.45 m). The flow rate was 0.5 mL/min. The linear
lL. The eluent was the mixture of 0.1 mol/L phos-
l
regression was calibrated with dextrans 80,000, 48,600, 25,000,
11,600, and 5,2000. Vt and V0 were calibrated with glucose and
dextran blue (2,000,000), respectively.
2.7. Monosaccharide analysis
The sample (2 mg) was dissolved in 2 mL of 2 mol/L trifluoro-
acetic acid (TFA) in a screw-capped vial, filled with nitrogen, and
hydrolyzed at 120 °C for 2 h. The hydrolyzate was evaporated with
a rotary evaporator. Neutral sugars and uronic acids were simulta-
neously detected by GC using the method described previously.9
GC was performed by a Shimadzu GC2010 equipped with a capil-
lary column of rtx-50 (30.0 m  0.25 mm  0.25 am). The temper-
ature program was: 180 °C for 2 min, then to 210 °C at 6 °C/min,
then to 215 °C at 0.3 °C/min, then to 240 °C at 6 °C/min for
30 min. Nitrogen was used as the carrier gas at 0.6 mL/min.
2.3. Isolation of cornus polysaccharides
The powder of the fruit of C. officinalis was extracted with water
(30 °C twice and then 80 °C once).7 The pectic polysaccharide FCP
was obtained as reported in our previous work.7 The remaining cell
debris after the water extraction was first extracted with 5% NaOH
at 4 °C for 4 h. After neutralization with 2 N HCl and centrifugation,
the debris was further extracted with 10% NaOH at 4 °C for another
4 h, then neutralized, and centrifuged. The two combined alkali ex-
tracts were pooled and concentrated with a rotary evaporator at a
temperature below 40 °C. A fourfold volume of ethanol was added
to the supernatant to precipitate the polysaccharides overnight at
4 °C. The precipitate was pelleted by centrifugation and re-dis-
solved in distilled H2O. The aqueous solution was treated with Se-
vag reagent to remove protein and then was dialyzed against
distilled water for 48 h, concentrated under reduced pressure,
and lyophilized. A black crude polysaccharide, CAP, was then
obtained.
2.8. Partial acid hydrolysis
FCAP1 (25 mg) was dissolved in 10 mL of 0.1 mol/L TFA and
heated at 100 °C for 2 h. The solution was evaporated to dryness,
then re-dissolved in distilled water and dialyzed for 48 h. Both dia-
lyzable and non-dialyzable samples were lyophilized. The non-dia-
lyzable fraction was further purified on a Sephadex G-25 column,
giving the partially hydrolyzed polysaccharide FCAP1-I; the dialyz-
able oligomers were freeze-dried and designated as FCAP1-O. Su-
gar compositions of FCAP1-I and FCAP1-O were analyzed by GC.
The structure of FCAP1-I was further analyzed by methylation.
2.4. Decoloring
2.9. Methylation analysis
CAP was dissolved in distilled water at 40 °C with continuous
stirring and the solution was adjusted to pH 8.8 with aqueous
ammonia. H2O2 (30%) was then added drop-wise until the color
faded. After stirring for another 4 h, the color turned primrose yel-
low. The solution was neutralized with 1 N HCl, followed by dialy-
sis against distilled water and lyophilization.
Both the samples of FCAP1 and FCAP1-I were methylated using
modified Ciucanu method as described previously.10 The methyla-
tion procedure was repeated three times. Next, the samples were
hydrolyzed with 2 mol/L TFA (121 °C, 2 h), then reduced with so-
dium borohydride, and acetylated to convert into their partially
methylated alditol acetates. The resulting alditol acetates were
analyzed by GC and GC–MS. GC–MS was performed using a Shima-
dzu instrument GCMS-QP2010 equipped with an electron impact
ion source (ionization energy 70 eV). The capillary column used
2.5. Purification of FCAP1
Decolored CAP (373 mg) was dissolved in water and fraction-
ated on DEAE–Cellulose DE-52 anion-exchange column
was rtx-5 ms (30 m  0.25 mm  0.25
lm); the temperature pro-
À
(5.0 cm  40 cm, HCO3 form), eluted first with distilled water,
gram was: 140 °C for 2 min, then to 250 °C at 2 °C/min for
20 min. Helium was used as a carrier gas and the flow rate was
0.6 mL/min. The temperatures of the interface and the ion source
were 200 °C and 250 °C, respectively.
and then with a step gradient (0.1, 0.25, and 0.5 mol/L NaHCO3)
at a flow rate of 1 mL/min, monitored spectrophotometrically at
485 nm using the H2SO4–phenol method. After pooling, dialysis,
and lyophilization, the fractions were designated as CAP1 (yields
25.20%), CAP2 (yields 6.43%), CAP3 (yields 11.53%), and CAP4
(yields 14.75%) separately. CAP1 (60 mg) was further purified with
gel permeation chromatography on a Bio-Gel P-30 column using
0.1 mol/L NaCl solution as an eluent. One fraction was pooled
according to the elution profile. After dialysis and lyophilization,
a white fluffy polysaccharide FCAP1 (yields 73.33%) was obtained.
2.10. Enzymatic hydrolysis
FCAP1 (7 mg) was dissolved in sodium acetate buffer (50 mM,
pH 5.0) containing 40 unit of endo-b-(1?4)-D-glucanase (EC
3.2.1.4 from Aspergillus niger). The solution was incubated at
37 °C for 48 h. The digest was precipitated with four volumes of
anhydrous ethanol and centrifuged. The resulting supernatant
was concentrated and lyophilized giving a mixture of oligosaccha-
rides P1-XGose. A glucanase-resistant fraction, designated as P1-
GRF, was obtained by lyophilization of ethanol-insoluble residues.
Monosaccharide composition of P1-XGose and P1-GRF was ana-
lyzed by GC. P1-XGose was further analyzed by MALDI-TOF mass
spectrum.
2.6. Homogeneity and molecular weight determination
The homogeneity and molecular weight of FCAP1 were deter-
mined by high performance gel permeation chromatography
(HPGPC) on TSK-Gel G3000SW column (7.5 mm  300 mm). A
Waters 2695 high performance liquid chromatography (HPLC)

L. Yang et al. / Carbohydrate Research 345 (2010) 1909–1913
1911
3. Results and discussion
3.1. Isolation and purification of cornus polysaccharides
Extraction of the cellular debris of C. officinalis with alkali
yielded a crude polysaccharide, CAP. The carbohydrate content of
CAP was 85.3% according to the H2SO4–phenol method. Monosac-
charide analysis by GC showed that it was composed of fucose,
arabinose, xylose, mannose, glucose, galactose, and trace of rham-
nose in a molar ratio of 0.20:0.66:2.12:1:2.37:0.98.
CAP was a black polysaccharide in that the pigment could not be
separated by column chromatography. To avoid the influence of pig-
ment during the structure analysis, decoloring was performed with
30% H2O2. Decolored CAP was purified by anion exchange chroma-
tography, yielding four polysaccharide subfractions CAP1, CAP2,
CAP3, and CAP4 (Fig. 1). CAP1 was the dominant sub-fraction
accounting for 44.3% of the total polymers recovered from the anion
exchanger. CAP1 was further purified by gel permeation chromatog-
raphy on a Bio-Gel P-30 column. As shown in Figure 2, one sub-frac-
tion, FCAP1, was obtained that accounted for 0.29% of the crude herb.
Figure 2. Elution profile of CAP1 on a Bio-Gel P-30 column. FCAP1 fraction was
obtained by eluting with 0.1 mol/L NaCl solution.
3.3. Homogeneity and molecular weight
3.2. Monosaccharide composition and character of FACP1
The homogeneity and molecular weight of FCAP1 were deter-
mined by high performance gel permeation chromatography
(HPGPC). FCAP1 was eluted with a mixture of 0.1 mol/L phosphate
buffer (pH 6.0) and 0.1 mol/L Na2SO4. The single symmetrical peak
indicated that the polymer was homogeneous. Based on the cali-
bration with standard dextrans, the average molecular weight
(MW) of the sample was estimated to be 34.5 kDa.
FCAP1 was a water-soluble, white, and fluffy polysaccharide. Its
monosaccharide composition is summarized in Table 1. FCAP1
mainly consisted of fucose, arabinose, xylose, mannose, glucose,
and galactose in a molar ratio of 0.29:0.19:1.74:1:3.30:1.10, and
uronic acid was not detected. As determined by a colorimetric
method, the total carbohydrate content was 91.3%, and protein
was absent (0%). A UV scan in the region of 200–400 nm showed
strong absorbance at about 200 nm but no distinct absorbance at
280 nm, which further indicated that the polysaccharide did not
contain protein.
3.4. Partial acid hydrolysis
FCAP1 was partially hydrolyzed with 0.1 mol/L TFA. The dialyz-
able fraction FCAP1-O contained fucose, arabinose, xylose, man-
nose, glucose, and galactose (Table 1), indicating that they were
present on the outer chains. The non-dialyzable fraction was fur-
ther fractionated on a Sephadex G-25 column and a sub-fraction
FCAP1-I was obtained. Based on HPGPC, its molecular weight was
16.6 kDa. FCAP1-I was composed of xylose, glucose, and galactose
in a molar ratio of 4.6:9.6:1 (Table 1). The monosaccharide compo-
sition of FCAP1-I indicated that xylose, glucose, and galactose were
present as the main chain or brink of the main chain. FCAP1-I had
no arabinose, fucose, or mannose suggesting that all arabinose, fu-
cose, and mannose were present on the branches of the
polysaccharide.
In the FT-IR spectrum, signals in the regions of 3600–3200 cmÀ1
,
2926 cmÀ1, 1642 cmÀ1 were due to O–H stretching vibration, C–H
stretching vibration, and associated water, respectively. The weak
absorbance at 895 cmÀ1 suggested that pyranoses existed in the b-
configuration.11 The IR spectrum showed no specific signature of
uronic acid in consistence with the data of GC analysis.
3.5. Methylation analysis
Methylation of FCAP1 and FCAP1-I was performed three times
to obtain fully methylated polysaccharides. After hydrolysis and
alditol acetate derivatization, the partially methylated alditol ace-
tates were analyzed by GC and GC–MS. Gas chromatography of
FCAP1 and FCAP1-I is shown in Figure 3. Peaks of methylated sug-
ars were identified by their retention times and mass spectra. Their
relative molar ratios were estimated from the peak areas of GC and
corresponding response factors.12 According to their molecular
weight and monosaccharide composition, the total molar ratio of
FCAP1 was 2.1 times higher than that of FCAP1-I. The results are
shown in Table 2.
Data from the methylation analysis indicated that FCAP1 had a
highly branched structure. The amount of total terminal sugars
was approximately equal to that of branched sugars, indicating
the completeness of methylation. The non-reducing termini were
composed of arabinose, xylose, fucose, and galactose. Branches
were present on glucosyl residues and on a trace of mannosyl res-
Figure 1. Elution profile of the decolored polysaccharide CAP from Cornus officinalis
on a DEAE–cellulose column (HCO3 form). CAP1, CAP2, CAP3, and CAP4 were
obtained by eluting with H2O, 0.1, 0.25, and 0.5 mol/L NaHCO3, respectively. A485
was determined by phenol–sulfuric acid method.
À

1912
L. Yang et al. / Carbohydrate Research 345 (2010) 1909–1913
Table 1
Sugar composition of CAP, FCAP1, and their derivatives from partial hydrolysis with acid and enzymatic hydrolysis
Polysaccharide fraction
Sugar composition (%)
Xylose
Rhamnose
Fucose
Arabinose
Mannose
Glucose
Galactose
CAP
FCAP1
FCAP1-I
FCAP1-O
P1-XGose
P1-GRF
0.8
—
—
—
—
2.7
3.8
—
8.2
3.7
1.5
8.9
2.5
—
5.0
2.1
4.1
28.7
22.8
30.5
19.7
19.4
27.9
13.6
13.1
—
19.5
12.4
17.4
32.1
43.4
62.9
26.3
46.1
36.5
13.2
14.4
6.6
21.3
16.3
12.6
—
—: Not detected.
(1?2)-linked galactose. The fucosyl residues were present at the
non-reducing termini. In addition, the mannosyl residues were
composed of (1?4)-linked and (1?4),6-linked mannose, which
suggested that mannose was probably existed as (1?4)-linked
mannose, with branches at O-6.
FCAP1-I showed only five peaks by GC, which were identified as
2,3,4-Me3-Xyl, 3,4-Me2-Xyl, 2,3,4,6-Me4-Gal, 2,3,6-Me3-Glc, and
2,3-Me2-Glc. Termini were composed of xylose and galactose.
2,3,6-Me3-Glc and 2,3-Me2-Glc accounted for 65% of total methyl-
ated sugars of FCAP1-I, which indicated that the backbone of
FCAP1-I was composed of (1?4)-linked-Glc, with branches at-
tached to O-6 of some residues. The amount of (1?2)-linked-Xyl
was equal to that of terminal galactose. It can be deduced from
the results that the structure of FCAP1-I was as follows (Fig. 4).
Compared with FCAP1, FCAP1-I had no terminal Ara, (1?3)-
linked-Ara, terminal Fuc, (1?4)-linked-Man, (1?4,6)-linked-
Man, and (1?2)-linked-Gal, suggesting that they existed as
branches. The number of terminal Xyl did not change, while the
number of (1?2)-linked-Xyl, terminal Gal, (1?4)-linked-Glc, and
(1?4,6)-linked-Glc was decreased. The decrease of (1?2)-linked-
Xyl, terminal Gal, and (1?4,6)-linked-Glc was probably due to
Gal-(1?2)-Xylp(1? substituted at O-6 of glucose was hydrolyzed
with 0.1 mol/L TFA, thus (1?4,6)-linked-Glc converted to (1?4)-
linked-Glc. The decrease of (1?4)-linked-Glc indicated that some
(1?4)-linked-Glc was located at branches.
Figure 3. Gas chromatography of partially methylated alditol acetates of FCAP1 and
m); temperature: 140 °C
(2 min)–2 °C/min, 250 °C (20 min). (1) 2,3,5-Me3-Ara; (2) 2,3,4-Me3-Xyl; (3) 2,3,4-
Me3-Fuc; (4) 2,5-Me2-Ara; (5) 3,4-Me2-Xyl; (6) 2,3,4,6-Me4-Gal; (7) 2,3,6-Me3-Man;
(8) 2,3,6-Me3-Glc; (9) 3,4,6-Me3-Gal; (10) 2,3-Me2-Man; (11) 2,3-Me2-Glc.
FCAP1-I. Column: rtx-5 ms (30 m  0.25 mm  0.25
l
From the analysis above, it can be concluded that FCAP1 is a
branched polysaccharide. The backbone is composed of b-(1?4)-
linked-glucose, with some substitution at O-6 position. Xylose is
linked directly to the backbone. The branches are composed of
(1?3)-linked-Ara, (1?4)-linked-Man, (1?4,6)-linked-Man, (1?4)-
linked-Glc, and (1?2)-linked-Gal. Arabinose, fucose, and galactose
are at the terminal of the branches.
Table 2
The results of methylation analysis of FCAP1 and FCAP1-I
Peak number
Methylated sugar
Deduced linkage
Molar ratio
FCAP1 FCAP1-I
3.6. Enzymatic hydrolysis
1
2
3
4
5
6
7
8
9
2,3,5-Me3-Ara
2,3,4-Me3-Xyl
2,3,4-Me3-Fuc
2,5-Me2-Ara
Ara-(1-
Xyl-(1-
Fuc-(1-
2
10
4
1
8
10
9
22
6
4
24
—
10
—
—
3
It is known that endo-b-(1?4)-D-glucanase cleaves (1?4)-b-
glucosidic linkages of xyloglucan next to an unbranched glucose
-3)-Ara-(1-
-2)-Xyl-(1-
Gal-(1-
-4)-Man-(1-
-4)-Glc-(1-
-2)-Gal-(1-
-4,6)-Man-(1-
-4,6)-Glc-(1-
residue without damaging the side chains.13 FCAP1 was treated
3,4-Me2-Xyl
with an endo-b-(1?4)-D-glucanase and oligomers were generated,
2,3,4,6-Me4-Gal
2,3,6-Me3-Man
2,3,6-Me3-Glc
3,4,6-Me3-Gal
2,3-Me2-Man
2,3-Me2-Glc
3
further indicating that FCAP1 had a backbone of b-(1?4)-linked-
glucose. The hydrolysate was precipitated with ethanol and the
supernatant, a mixture of oligosaccharides, was designated as P1-
XGose, which was a mixture of oligosaccharides generated from
the enzymatic hydrolysis. The precipitate fraction, which was the
—
17
—
—
14
10
11
—: Not detected.
idues. 2,3,6-Me3-Glc and 2,3-Me2-Glc accounted for 46% of total
methylated sugars suggesting that the backbone was composed
of (1?4)-linked-Glc, with substitutions at O-6 of glucose. The xylo-
syl residues were composed of 2,3,4-Me3-Xyl and 3,4-Me2-Xyl res-
idues, which suggested that xylose was present in the pyranose
form. The galactosyl residues were composed of terminal- and
Figure 4. Structural unit of FCAP1-I.

L. Yang et al. / Carbohydrate Research 345 (2010) 1909–1913
1913
Figure 5. MALDI-TOF mass spectrum of oligosaccharides in P1-Xgose.
enzyme-resistant polymeric material, was designated as P1-GRF.
4. Conclusion
Sugar analysis of P1-XGose and P1-GRF is summarized in Table 1.
Xylose and glucose are the major sugars of the P1-XGose in the
ratio of 1:2.4. suggesting that FCAP1 is a polysaccharide rich in
Based on the methylation analysis, partial acid hydrolysis, enzy-
matic hydrolysis, GC–MS, and MALDI-TOF-MS analysis, we con-
xyloglucan that could be digested by endo-b-(1?4)-D-glucanase.
clude that FCAP1 is
a highly branched xyloglucan with a
The presence of fucose and galactose in P1-XGose suggested that
xyloglucan had fucose and galactose-containing side chains. Man-
nose was present in P1-XGose. It was likely that the endo-b-(1?4)-
backbone of b-(1?4)-linked-glucopyranose residues. The back-
bone was partially substituted at O-6 of glucosyl residues with
Xylp-(1?, Galp-(1?2)-Xylp-(1? and Fucp-(1?2)-Galp-(1?2)-
Xylp-(1?. The branches also contained ?3)-Ara-(1?4)-Glc-
(1?4)-Man(1?4,6)-Man-(1?, Ara-(1? and Gal-(1?. Herein we
also report, for the first time, the degradation of xyloglucan from
D
-glucanase contains contaminating mannanase activity responsi-
ble for mannan hydrolysis.14 The enzyme-resistant fraction P1-
GRF had a similar sugar composition as FCAP1. The presence of xy-
lose and glucose indicated that a part of the xyloglucan was resis-
C. officinalis with endo-b-(1?4)-D-glucanase.
tant to endo-b-(1?4)-D-glucanase.
MALDI-TOF-mass spectrometry is a convenient tool for the
structural analysis of highly branched xyloglucan mixtures as it
is sensitive in mixture analysis. The MALDI-TOF mass spectrum
of P1-XGose is shown in Figure 5. As shown in the figure, the mass
spectrum of P1-XGose revealed numerous peaks of oligosaccharide
fragments. Structures of the xyloglucan-derived oligomers were
proposed, taking into consideration the specificity and mode of ac-
Acknowledgments
Financial support from the Program for New Century Excellent
Talents in Universities (NCET-08-0893), the Hi-Tech Research and
Development Program of China (Nos. 2007AA10Z338, 2007AA
091601), and Shaanxi Provincial Science Development Program
(2007k16-06(3)) is gratefully acknowledged.
tion of the endo-b-(1?4)-D-glucanase, sugar composition and gly-
cosyl linkage data, and molecular masses of the known xyloglucan
oligosaccharides.
Ions at m/z 1247.74, 1409.47, and 1555.53, corresponding,
respectively, to [M+Na]+ adducts of Hex5Pent3, Hex6Pent3, and
Hex6Pent3dHex, can be assigned as XXLG (or XLXG), XLLG, and
XLFG fragments of the XXXG-type xyloglucan,15 respectively, (X:
References
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Arts Institute of USA, 1982. p 19.
2. Zhou, J. H.; Li, C. S.; Li, D. D. Chin. J. New Drug 2001, 10, 808–811.
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11. Zhang, W. J. Techniques of Glycoconjugates in Biochemistry; Zhejiang University
Press: Hangzhou, 1994. pp 271–279.
12. Sweet, D. P.; Shapiro, R. H.; Albersheim, P. Carbohydr. Res. 1975, 40, 217–225.
13. Fry, S. C. J. Exp. Bot. 1989, 40, 1–11.
14. Mazumder, S.; Lerouge, P.; Loutelier-Bourhis, C.; Driouich, A.; Ray, B. Carbohydr.
Polym. 2005, 59, 231–238.
a
D
-
D
-Xylp-(1?6)-b-
-Glcp; G: b- -Glcp; F:
(1?6)-b- -Glcp). The high intensity of XLFG indicated that the
D
-Glcp; L: b-
D
-Galp-(1?2)-
a
-
D
-Xylp-(1?6)-b-
D
a
-L
-Fucp-(1?2)-b-
D
-Galp-(1?2)-
a-D-Xylp-
D
xyloglucan was fucosylated. Ions at m/z 821.31, 953.35, 1099.41,
and 1261.46, corresponding, respectively, to [M+Na]+ adducts of
Hex4Pent, Hex4Pent2, Hex4Pent2dHex, and Hex5Pent2dHex, can be
assigned as LGG, XLG, XFG, and LFG, respectively. These structures
can be ascribed to the XXG-type xyloglucan.16 In addition, sodium
adduct ions of hexose polymers ranging from four to seven (m/z
689.39, 851.42, 1013.51, and 1175.56) were also observed. P1-
XGose contain high amount of hexose residues of mannose, galact-
ose, and glucose, and the branches of FCAP1 contain abundant
mannose, galactose, and glucose. Thus these peaks probably origi-
nated from oligosaccharides on side chains composed of mannan,
galactose, and glucose.
15. Huisman, M. M. H.; Weel, K. G. C.; Schols, H. A.; Voragen, A. G. J. Carbohydr.
Polym. 2000, 42, 185–191.
16. Hilz, H.; Jone, L. E.; Kabel, M. A.; Verhoef, R.; Schols, H. A.; Voragen, A. G. J.
Carbohydr. Res. 2007, 342, 170–181.